The major focus of the project is to elucidate mechanisms controlling cell fate decisions in developing T cells. Early precursor thymocytes that commit to the T cell lineage must specify an alpha-beta or gamma-delta T cell fate. Precursor T cells developing in the alpha-beta T cell pathway undergo a testing process in the thymus to ensure that cells expressing useless or self-reactive TCR do not mature (positive and negative selection). These selection processes require TCR engagement of self-MHC antigens, but the nature of these interactions determine whether the cell will mature or die. Depending on the class of MHC recognized, maturing thymocytes will adopt a CD4 or CD8 T cell fate and the associated effector functions characteristic of these lineages. A major goal in our work is to understand how TCR signals, acting in concert with other developmental cues, are linked to processes of lineage commitment. In previous studies, we have shown that quantitative differences in TCR signaling influence cell fate decisions in developing T cells. Deficiencies in specific kinases required for the activation of TCR, can alter lineage choice, promoting CD8 development of MHC Class II-specific thymocytes that would normally specify the CD4 fate. Likewise, decreasing competition for MHC promotes the development of cells with sub-optimal Class II recognition to develop in the CD8 pathway. Even limiting thymocyte TCR-Class II MHC interactions with non-selectable and dying thymocyte neighbors can redirect lineage commitment. Since such an environment limits MHC access, not only quantity, but also the kinetics of TCR signaling must play a role in lineage commitment, perhaps by affecting the integration of TCR signals over time and space. Of note, we find that a constitutively active form of Notch can override the bias normally imposed by specific TCR signals, suggesting that Notch and the TCR may act in concert to specify cell fate. In efforts to understand how Notch signaling is regulated in the thymus, we have manipulated the expression of Presenilins (PS1/2), the proteins required for generating the active form of Notch. We have generated mutant mice for assessing Notch function in vivo that circumvent the problems of redundancy in Notch receptors/ligands, early lethality associated with deletion mutants, and those associated with ectopic and over expression of transgenes. Transgenic constructs, expressing a dominant negative form of presenilin, produce a profound block in T cell development while promoting ectopic development of B cells. This protein operates in early thymic precursors, enhancing NK development while attenuating gamma-delta development. Dominant negative Presenilin appears to mediate it effects through Notch since the functional defects imposed by the transgene can be compensated by co-expression of a transgene encoding the actived form of Notch. In other models, we have generated conditional null mutations of PS1/2, targeting gene deletion to specific stages of thymocyte development. In one of these, we find alterations in TCR signaling at the stage of alpha-beta development where selection occurs and, unexpectedly, the maturation of CD4 T cells is partially inhibited. Collectively, these studies indicate that both TCR and Notch influence cell fate decisions at several stages of thymic development, including T/B lineage commitment.